PROJECT SUMMARY Arsenic-induced cancers of lung, skin, liver, and bladder pose serious health threats given the global extent of environmental arsenic contamination. Although the mechanism by which arsenic drives carcinogenesis is still poorly understood, increasing evidence implicates reactive oxygen species (ROS) in transformation of cells to a malignant phenotype. NF-E2-related factor-2 (Nrf2) is a key transcription factor that regulates antioxidant proteins to neutralize ROS and restore cellular redox balance. Indeed, many natural compounds with anticancer properties exhibit protective effects by activating Nrf2. Conversely, constitutive activation of Nrf2, which is prominent in several types of human cancer cell lines and tumors, is known to be oncogenic, as it protects cancer cells against oxidative stress and chemotherapeutics. Our preliminary studies have shown that human lung bronchial epithelial (BEAS-2B) cells exposed to arsenic generate ROS, leading to malignant cell transformation and a cascade of subsequent effects: sharply reduced level of ROS; increased levels of antioxidants; constitutive activation of Nrf2; and resistance to apoptosis. These arsenic-transformed cells also exhibit autophagy impairment, leading to accumulation of p62 protein and constitutive activation of Nrf2, high levels of inflammatory agents COX-2 and TNF-?, and activation of transcription factor hypoxia-inducible factor (HIF)-1?. Animals exposed to arsenic through drinking water showed both activated HIF-1? and Nrf2. Knockdown of Nrf2 down-regulates HIF-1?, resulting in decreased angiogenesis of arsenic-transformed cells. The central hypothesis is that inducible Nrf2 is anti-oncogenic at the early stage of arsenic-induced carcinogenesis (cell transformation) via up-regulation of antioxidants to decrease ROS while constitutively expressed Nrf2 is oncogenic in the later stages (tumorigenesis and metastasis) by inducing resistance to apoptosis, creating an inflammatory microenvironment, and activating angiogenesis. Aim 1 will elucidate a protective role of inducible Nrf2 in arsenic-induced malignant cell transformation, decrease of ROS by Nrf2, induction of Nrf2 target antioxidant proteins by arsenic, and protective role of Nrf2 in arsenic-induced oxidative damage. Aim 2 will establish the oncogenic role of constitutively activated Nrf2 in development of resistance to apoptosis of arsenic-transformed cells. The study will determine whether p62 is a positive inducer of Nrf2, establish a mechanism for Bcl-2 up-regulation, and elucidate the roles of decreased ROS generation and Bcl-2 up-regulation in apoptosis resistance. Aim 3 will establish the oncogenic role of constitutive Nrf2 in tumorigenesis and metastasis of arsenic-transformed cells in vivo by deciphering the role of p62/Nrf2 in creating an inflammatory microenvironment by studying activation of NF-?B and expressions of COX-2 and TNF-? in arsenic-transformed cells. The roles of p62, Nrf2, inflammatory proteins, angiogenesis, and apoptosis resistance in arsenic-induced tumorigenesis and metastasis will be investigated using animal models.